Hepatic insulin resistance due to obesity is central to the pathogenesis of common disorders including non- alcoholic fatty liver disease (NAFLD). This research proposal addresses the unanswered question of whether membrane phospholipid composition regulates hepatic insulin sensitivity. Our long-term goal is to understand mechanistic relationships between membrane phospholipids and metabolic regulation, particularly where they present therapeutic opportunities. The objective of this research is to understand a fundamental new hepatocellular mechanism for the regulation of lipid and glucose homeostasis. The central hypothesis is that, under physiological conditions, phosphatidylcholine transfer protein (PC-TP) functions as an intracellular sensor of membrane phospholipid composition that limits insulin signaling by binding thioesterase superfamily member 2 (Them2) and tuberous sclerosis complex 2 (TSC2). In the setting of overnutrition and obesity, we postulate that PC-TP-mediated activation of Them2 promotes hepatic insulin resistance due to overproduction of saturated free fatty acids, which in turn promote endoplasmic reticulum (ER) stress. The rationale is that the mechanisms of PC-TP-mediated metabolic regulation should yield new insights into hepatic insulin resistance. Guided by extensive preliminary data, the central hypothesis will be tested in three specific aims: 1) To determine the mechanisms by which PC-TP and Them2 regulate insulin signaling; 2) To ascertain the pathogenic roles of PC-TP and Them2 in ER stress; and 3) To define roles of phosphatidylcholines in the activation of Them2 and TSC2 by PC-TP. In Aim 1, a newly developed small molecule inhibitor of PC-TP, as well as Pctp-/- and Them2-/- mice and cultured hepatocytes will be used to examine whether a complex of PC-TP and Them2 limits insulin signaling and thereby promotes hepatic glucose production and triglyceride synthesis under fasting conditions. Aim 2 will utilize high fat fed Pct-/- and Them2-/- mice, as well as leptin-deficient ob/ob mice to establish the pathogenic roles of PC-TP and Them2 in ER stress. Mice, cultured hepatocytes and Hepa 1-6 mouse hepatoma cells will be used to evaluate whether PC-TP, via the activity of Them2, exacerbates free fatty acid-induced depletion of calcium from the ER lumen. In Aim 3, complementary approaches will be used to examine whether nutritional status influences the molecular species of phosphatidylcholines bound to PC-TP and whether the bound phosphatidylcholines regulate PC-TP's interactions with Them2 and TSC2. Phosphatidylcholine-dependent interactions will be quantified by pulldown assays, surface plasmon resonance, and the activities of Them2 and TSC2, as well as by a mammalian two-hybrid assay system. Overall, this proposal will elucidate mechanisms of PC-TP-mediated regulation of hepatic insulin sensitivity, which are significant because hepatic phosphatidylcholine composition varies in health and disease. These studies are expected to identify new therapeutic targets for the management of hepatic insulin resistance and related disorders.